Investigation of acid phosphatase variants for the synthesis of phosphate monoesters

Abstract

The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;114: 2187-2195. © 2017 Wiley Periodicals, Inc.

Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters

“…The product concentrations were in the same range as those obtained at pH 4.2 and could be maintained over at least 1 d. Under these conditions, AcP was completely consumed when the product plateau was reached and no bis‐phosphorylated product was observed. Similar observations were made with acid phosphatases and PP i as donor,31,33,35,37 however, high product levels could not generally be maintained, if any, at the cost of large P i side‐product formation. A difference between the pH optimum of phosphotransferase and phosphohydrolase activities has been also observed with calf intestine alkaline phosphatase 30…”

“…BSAR‐1,47 PhoC−Mm wild‐type and G92D/I171T from Morganella morganii 35 and NSAP−Eb 11‐variant from Escherichia blattae 35 were expressed as reported. Phytase from Aspergillus niger was from BASF.…”

Preparative‐Scale Enzymatic Synthesis of rac‐Glycerol‐1‐phosphate from Crude Glycerol Using Acid Phosphatases and Phosphate

“…Encouraged by these previous results, we initiated our study by testing several nonspecific acid phosphatases (NSAPs), which included PhoN‐Sf from Shigella flexneri , PhoN‐Se from Salmonella typhimurium LT2, PiACP from Prevotella intermedia , Lw from Leptotrichia wadei , and PhoC‐Mm from Morganella morganii , as well as a histidine acid phosphatase and a phytase from Aspergillus niger for the regioselective synthesis of glycerol‐1‐phosphate. High concentrations of glycerol [pure glycerol ( 1 a ) and crude glycerol ( 1 b )] and PP i as the P‐donor were employed in overnight reactions at 30 °C in water.…”

“…Acommon drawback of phosphatase-catalyzed transphosphorylation reactions lies in the undesired enzyme-catalyzed hydrolysis of the newly formed products.I na ddition to the use of high loadings of both alcohols ubstrate and P-donor, [19][20][21][22] varioust echniques have been developed to minimize product hydrolysis, such as continuous-flows ynthesis, [23,24] enzymee ngineering, [25,26] and reaction engineering. [27] They could enable the mild and selective enzymatic synthesis of phosphate monoesters of functionalized alcohols and sugars.…”

“…In contrast, phytases could utilize 20 wt % NaH 2 PO 4 as the monophosphate salt for the direct condensation in 64 wt % glycerol to furnish 104 g L −1 product. In comparison, calf intestine alkaline phosphatase, which also delivers approximately 10 % of glycerol‐2‐phosphate in the phosphorylation of glycerol, could deliver 71 g L −1 product at best, whereas the product titer with nonspecific acid phosphatases reached only approximately 17 g L −1 from pyrophosphate under standard conditions . Importantly, several enzymes could operate efficiently in crude glycerol obtained from biodiesel production.…”